Project Summary 53BP1 is essential for non-homologous end-joining (NHEJ) of DNA lesions and is tightly regulated by reversible phosphorylation. We recently reported that dephosphorylation of 53BP1 at residues T1609 and S1618 in its foci-forming region (FFR), catalyzed by the PP4C-PP4R3? phosphatase complex, is necessary for its recruitment to double-strand breaks (DSB) during G1. Independent lines of evidence suggest that dephosphorylation-dependent recruitment of 53BP1 to DSB could be leveraged in the context of cancer therapy. Mutations in BRCA1, which are frequently found in breast and ovarian cancers, compromise HR and render these tumors exquisitely sensitive to PARP inhibitors. The response to PARP inhibitors is strongly dependent on the function of 53BP1. In fact, depletion of 53BP1 in BRCA1-mutant cells restores homologous recombination (HR) proficiency and renders these cells resistant to PARP inhibitors. Secondly, a mutation in 53BP1 within the sequence spanningT1609/S1618 was identified in a breast cancer patient and we found that this mutation disrupts 53BP1 recruitment to DNA damage foci, and induces resistance to PARP inhibitors. Importantly, the functional deficiency in 53BP1 (deletion or mutation) enhances radiosensitivity. Therefore BRCA1-mutant tumors that develop resistance to PARP inhibitors due to loss in 53BP1 function are likely to respond to radiotherapy. Based on these results we hypothesize that the PP4C-53BP1 axis has significant therapeutic implications specifically in BRCA1-mutant tumors. We have observed that phosphorylation of the Ser840 residue in a fragment in the C-terminus of PP4R3? is necessary for the formation of PP4R3?/53BP1 complex. In Aim 1 we will utilize phosphoproteomic methods to examine the specificity of PP4C/PP4R3? mediated 53BP1 dephosphorylation. Use in vitro binding assays and cell based assays to determine whether the interaction of PP4R3? and 53BP1 is direct, or mediated by other factors. Finally use time-lapse imaging to examine the kinetics of interaction of PP4R3? and 53BP1 during late mitosis and early G1. Our preliminary results suggest that Cdk5 mediated phosphorylation of the Ser840 residue on PP4R3? is critical for the interaction of PP4R3? and 53BP1. Aim 2 will utilize several innovative chemical genetic tools to systematically investigate the connection of Cdk5 with the PP4/53BP1 axis. Using an extremely specific and potent Cdk5 inhibitor, we will determine the precise impact of Cdk5 on PP4R3? phosphorylation during mitosis. 53BP1 foci formation in G1, and broadly assess its impact on DSB repair. Furthermore proteomics coupled to an unbiased chemical genetic approach will allow us to identify other Cdk5 substrates involved in DNA repair in cells. In Aim 3 we will identify and investigate the impact of sporadic mutations in the FFR of 53BP1, a PP4R3?-S840F mutation and inhibition of Cdk5 on olaparib sensitivity of BRCA1-mutant ovarian and breast tumor lines in vitro and for selected ones in orthotopically implanted mouse models. We will test whether olaparib-resistance can be overcome by radiation therapy using a sophisticated a small animal irradiator.